I. Function and Regulation of Intestinal Tight Junctions
The intestinal epithelium represents the largest interface (more than 2,000,000 cm2) between the external environment and the internal milieu. The maintenance of intercellular tight junctions (“tj”) competence prevents movements of potentially harmful environmental factors, such as bacteria, viruses, toxins, food allergens, and macromolecules across the intestinal barrier. This competence is significantly jeopardized in a variety of clinical conditions affecting the gastrointestinal tract, including food allergies, enteric infections, malabsorption syndromes, and inflammatory bowel diseases.
The tj or zonula occludens (hereinafter “ZO”) are one of the hallmarks of absorptive and secretory epithelia (Madara, J. Clin. Invest., 83:1089–1094 (1989); and Madara, Textbook of Secretory Diarrhea Eds. Lebenthal et al, Chapter 11, pages 125–138 (1990)). As a barrier between apical and basolateral compartments, they selectively regulate the passive diffusion of ions and water-soluble solutes through the paracellular pathway (Gumbiner, Am. J. Physiol., 253 (Cell Physiol. 22):C749–C758 (1987)). This barrier maintains any gradient generated by the activity of pathways associated with the transcellular route (Diamond, Physiologist, 20:10–18 (1977)).
Variations in transepithelial conductance can usually be attributed to changes in the permeability of the paracellular pathway, since the resistances of enterocyte plasma membranes are relatively high (Madara (1989, 1990), supra). The ZO represents the major barrier in this paracellular pathway, and the electrical resistance of epithelial tissues seems to depend on the number of transmembrane protein strands, and their complexity in the ZO, as observed by freeze-fracture electron microscopy (Madara et al, J. Cell Biol., 101:2124–2133 (1985)).
There is abundant evidence that ZO, once regarded as static structures, are in fact dynamic and readily adapt to a variety of developmental (Magnuson et al, Dev. Biol., 67:214–224 (1978); Revel et al, Cold Spring Harbor Symp. Quant. Biol., 40:443–455 (1976); and Schneeberger et al, J. Cell Sci., 32:307–324 (1978)), physiological (Gilula et al, Dev. Biol., 50:142–168 (1976); Madara et al, J. Membr. Biol., 100:149–164 (1987); Mazariegos et al, J. Cell Biol., 98:1865–1877 (1984); and Sardet et al, J. Cell Biol., 80:96–117 (1979)), and pathological (Milks et al, J. Cell Biol., 103:2729–2738 (1986); Nash et al, Lab. Invest., 59:531–537 (1988); and Shasby et al, Am. J. Physiol., 255(Cell Physiol., 24:C781–C788 (1988)) circumstances. The regulatory mechanisms that underlie this adaptation are still not completely understood. However, it is clear that, in the presence of Ca2+, assembly of the ZO is the result of cellular interactions that trigger a complex cascade is of biochemical events that ultimately lead to the formation and modulation of an organized network of ZO elements, the composition of which has been only partially characterized (Diamond, Physiologist, 20:10–18 (1977)). A candidate for the transmembrane protein strands, occluden, has recently been identified (Furuse et al, J. Membr. Biol., 87:141–150 (1985)).
Six proteins have been identified in a cytoplasmic submembranous plaque underlying membrane contacts, but their function remains to be established (Diamond, supra). ZO-1 and ZO-2 exist as a heterodimer (Gumbiner et al, Proc. Natl. Acad. Sci., USA, 88:3460–3464 (1991)) in a detergent-stable complex with an uncharacterized 130 kD protein (ZO-3). Most immunoelectron microscopic studies have localized ZO-1 to precisely beneath membrane contacts (Stevenson et al, Molec. Cell Biochem., 83:129–145 (1988)). Two other proteins, cingulin (Citi et al, Nature (London), 333:272–275 (1988)) and the 7H6 antigen (Zhong et al, J. Cell Biol., 120:477–483 (1993)) are localized further from the membrane and have not yet been cloned. Rab 13, a small GTP binding protein has also recently been localized to the junction region (Zahraoui et al, J. Cell Biol., 124:101–115 (1994)). Other small GTP-binding proteins are known to regulate the cortical cytoskeleton, i.e., rho regulates actin-membrane attachment in focal contacts (Ridley et al, Cell, 70:389–399 (1992)), and rac regulates growth factor-induced membrane ruffling (Ridley et al, Cell, 70:401–410 (1992)). Based on the analogy with the known functions of plaque proteins in the better characterized cell junctions, focal contacts (Guan et al, Nature, 358:690–692 (1992)), and adherens junctions (Tsukita et al, J. Cell Biol., 123:1049–1053 (1993)), it has been hypothesize that tj-associated plaque proteins are involved in transducing signals in both directions across the cell membrane, and in regulating links to the cortical actin cytoskeleton.
To meet the many diverse physiological and pathological challenges to which epithelia are subjected, the ZO must be capable of rapid and coordinated responses that require the presence of a complex regulatory system. The precise characterization of the mechanisms involved in the assembly and regulation of the ZO is an area of current active investigation.
There is now a body of evidence that tj structural and functional linkages exist between the actin cytoskeleton and the tj complex of absorptive cells (Gumbiner et al, supra; Madara et al, supra; and Drenchahn et al, J. Cell Biol., 107:1037–1048 (1988)). The actin cytoskeleton is composed of a complicated meshwork of microfilaments whose precise geometry is regulated by a large cadre of actin-binding proteins. An example of how the state of phosphorylation of an actin-binding protein might regulate cytoskeletal linking to the cell plasma membrane is the myristoylated alanine-rich C kinase substrate (hereinafter “MARCKS”). MARCKS is a specific protein kinase C (hereinafter “PKC”) substrate that is associated with the cytoplasmic face of the plasma membrane (Aderem, Elsevier Sci. Pub. (UK), pages 438–443 (1992)). In its non-phosphorylated form, MARCKS crosslinks to the membrane actin. Thus, it is likely that the actin meshwork associated with the membrane via MARCKS is relatively rigid (Hartwig et al, Nature, 356:618–622 (1992)). Activated PKC phosphorylates MARCKS, which is released from the membrane (Rosen et al, J. Exp. Med., 172:1211–1215 (1990); and Thelen et al, Nature, 351:320–322 (1991)). The actin linked to MARCKS is likely to be spatially separated from the membrane and be more plastic. When MARCKS is dephosphorylated, it returns to the membrane where it once again crosslinks actin (Hartwig et al, supra; and Thelen et al, supra). These data suggest that the F-actin network may be rearranged by a PKC-dependent phosphorylation process that involves actin-binding proteins (MARCKS being one of them).
A variety of intracellular mediators have been shown to alter tj function and/or structure. Tight junctions of amphibian gallbladder (Duffey et al, Nature, 204:451–452 (1981)), and both goldfish (Bakker et al, Am. J. Physiol., 246:G213–G217 (1984)) and flounder (Krasney et al, Fed. Proc., 42:1100 (1983)) intestine, display enhanced resistance to passive ion flow as intracellular cAMP is elevated. Also, exposure of amphibian gallbladder to Ca2+ ionophore appears to enhance tj resistance, and induce alterations in tj structure (Palant et al, Am. J. Physiol., 245:C203–C212 (1983)). Further, activation of PKC by phorbol esters increases paracellular permeability both in kidney (Ellis et al, C. Am. J. Physiol., 263 (Renal Fluid Electrolyte Physiol. 32): F293–F300 (1992)), and intestinal (Stenson et al, C. Am. J. Physiol., 265(Gastrointest. Liver Physiol., 28):G955–G962 (1993)) epithelial cell lines.
II. Zonula Occludens Toxin
Most Vibrio cholerae vaccine candidates constructed by deleting the ctxA gene encoding cholera toxin (CT) are able to elicit high antibody responses, but more than one-half of the vaccinees still develop mild diarrhea (Levine et al, Infect. Immun., 56(1):161–167 (1988)). Given the magnitude of the diarrhea induced in the absence of CT, it was hypothesized that V. cholerae produce other enterotoxigenic factors, which are still present in strains deleted of the ctxA sequence (Levine et al, supra). As a result, a second toxin, zonula occludens toxin (hereinafter “ZOT”) elaborated by V. cholerae and which contribute to the residual diarrhea, was discovered (Fasano et al, Proc. Natl. Acad. Sci., USA, 8:5242–5246 (1991)). The zot gene is located immediately adjacent to the ctx genes. The high percent concurrence of the zot gene with the ctx genes among V. cholerae strains (Johnson et al, J. Clin. Microb., 31/3:732–733 (1993); and Karasawa et al, FEBS Microbiology Letters, 106:143–146 (1993)) suggests a possible synergistic role of ZOT in the causation of acute dehydrating diarrhea typical of cholera. Recently, the zot gene has also been identified in other enteric pathogens (Tschape, 2nd Asian-Pacific Symposium on Typhoid fever and other Salomellosis, 47(Abstr.) (1994)).
It has been previously found that, when tested on rabbit ileal mucosa, ZOT increases the intestinal permeability by modulating the structure of intercellular tj (Fasano et al, supra). It has been found that as a consequence of modification of the paracellular pathway, the intestinal mucosa becomes more permeable. It also was found that ZOT does not affect Na+-glucose coupled active transport, is not cytotoxic, and fails to completely abolish the transepithelial resistance (Fasano et al, supra).
More recently, it has been found that ZOT is capable of reversibly opening tj in the intestinal mucosa, and thus ZOT, when co-administered with a therapeutic agent, e.g., insulin, is able to effect intestinal delivery of the therapeutic agent, when employed in an oral dosage composition for intestinal drug delivery, e.g., in the treatment of diabetes (WO 96/37196; U.S. Pat. No. 5,827,534; U.S. Pat. No. 5,665,389; and Fasano et al, J. Clin. Invest., 99:1158–1164 (1997); each of which is incorporated by reference herein in their entirety). It has also been found that ZOT is capable of reversibly opening tj in the nasal mucosa, and thus ZOT, when co-administered with a therapeutic agent, is able to enhance nasal absorption of a therapeutic agent (U.S. Pat. No. 5,908,825; which is incorporated by reference herein in its entirety).
In U.S. Pat. No. 5,864,014; which is incorporated by reference herein in its entirety, a ZOT receptor has been identified and purified from an intestinal cell line, i.e., CaCo2 cells. Further, in U.S. Pat. No. 5,912,323; which is incorporated by reference herein in its entirety, ZOT receptors from human intestinal, heart and brain tissue have been identified and purified. The ZOT receptors represent the first step of the paracellular pathway involved in the regulation of intestinal and nasal permeability.
III. Zonulin
In U.S. Pat. Nos. 5,945,510 and 5,948,629, which are incorporated by reference herein in their entirety, mammalian proteins that are immunologically and functionally related to ZOT, and that function as the physiological modulator of mammalian tight junctions, have been identified and purified. These mammalian proteins, referred to as “zonulin”, are useful for enhancing absorption of therapeutic agents across tj of intestinal and nasal mucosa, as well as across tj of the blood brain barrier.
IV. Peptide Antagonists of Zonulin
Peptide antagonists of zonulin were identified and described for the first time in pending U.S. patent application Ser. No. 09/127,815, filed Aug. 3, 1998, which is incorporated by reference herein in its entirety, which corresponds to WO 00/07609. Said peptide antagonists bind to the ZOT receptor, yet do not function to physiologically modulate the opening of mammalian tight junctions. The peptide antagonists competitively inhibit the binding of ZOT and zonulin to the ZOT receptor, thereby inhibiting the ability of ZOT and zonulin to physiologically modulate the opening of mammalian tight junctions.
V. Diabetes
The morbidity and mortality associated with diabetes is devastating. The total number of diabetic individuals in the United States is 15.7 million. Of these, 100% of the type I diabetic individuals and 40% of type II diabetic individuals depend on parenteral administration of insulin. On an annual basis, the direct medical costs associated s with diabetes exceeds 40 billion dollars. An additional 14 billion dollars is associated with disability, work loss, and premature mortality.
Although oral insulin drug delivery strategies have been the focus of many research efforts, they have been largely unsuccessful because the physiologic nature of the small intestine prevents the absorption of macromolecules, such as insulin.
An oral dosage composition comprising ZOT for targeting delivery of insulin to the paracellular pathway for the treatment of diabetes has been described in U.S. Pat. Nos. 5,827,534 and 5,665,389. By physiologically modulating the paracellular pathway using ZOT, it is now possible to introduce a wide variety of therapeutic agents into the systemic circulation. This drug delivery system adds targeting specificity, which has long hampered the design of many oral pharmaceutical agents. The utility of this system is not limited to insulin delivery, and may represent a new way of designing orally administered pharmaceutical agents.
While offering an innovative treatment strategy for a disease as debilitating as diabetes is promising, preventing or delaying the onset of disease has widespread implications. Understanding the pathogenesis of any disease process is a daunting task. Heretofore, there has been no prior evidence of a pharmaceutical agent with the capability of preventing or delaying the onset of diabetes. In the present invention new light has been shed on the pathogenesis, prevention and delaying of onset of diabetes by demonstrating that a critical and early step in disease progression resides in alterations in paracellular permeability. In the present invention, it has been demonstrated that an increase in paracellular permeability is necessary for the progression toward diabetes. Peptide antagonists of zonulin, which block this endogenous pathway, have been found in the present invention to prevent the progression to diabetes. Thus, the present invention is believed to be useful to prevent long-term complications of diabetes. Further, the permeability changes associated with autoimmune diseases are long standing, and early intervention per the present invention is believed to have untold benefits to the diabetic patient.